Functional Characterization of Saccharomyces cerevisiae P5C Reductase, the Enzyme at the Converging Point of Proline and Arginine Metabolism

Abstract

The enzyme that, in Saccharomyces cerevisiae, catalyzes the last step in both proline synthesis and arginine catabolism, d1-pyrroline-5-carboxylate (P5C) reductase, was purified to near homogeneity and characterized thoroughly. Retention patterns upon gel permeation chromatography were consistent with a homodecameric composition of the holomer. High lability of the purified preparations and stabilization by reducing compounds suggested susceptibility to reactive-oxygen-species-mediated damage. Both NADH and NADPH were used as the electron donor, the latter resulting in a 3-fold higher Vmax. However, a higher affinity toward NADH was evident, and the NADPH-dependent activity was inhibited by NAD+, proline, arginine, and a variety of anions. With proline and arginine, the inhibition was of the competitive type with respect to the specific substrate, and of the uncompetitive- or mixed-type with respect to NADPH, respectively. The results suggest that, contrary to the enzyme from higher plants, yeast P5C reductase may preferentially use NADH in vivo. An in silico analysis was also performed to investigate the structural basis of such enzyme features. Superposition of the protein model with the experimental structure of P5C reductase from Medicago truncatula allowed us to hypothesize on the possible allosteric sites for arginine and anion binding, and the cysteine pairs that may be involved in disulfide formation.

Improving Drought Resistance by Introduction of the Proline Synthesis Gene P5CSA into Inbred Rice Lines as a New Genetic Resource for Breeding Programs

“…Similar ndings occurred in Arabidopsis during drought stress (Chen et al 2018) and in common bean during salinity and drought stress (Arteaga et al 2020). An increase in P5C reductase in relation to proline accumulation has been reported in previous studies Forlani et al 2022). In Arabidopsis and rice, P5CS was overexpressed under high salinity and dehydration stress and simultaneous accumulation of proline (Arabia et al 2021).…”

Functional Impact of a Cancer-Related Variant in Human Δ1-Pyrroline-5-Carboxylate Reductase 1

“…Assays were also conducted under conditions designed to mimic physiological substrate concentrations, as that reported previously for P5C reductase from Saccharomyces cerevisiae . 42 These assays were performed at 23 °C in phosphate-buffered saline (PBS) solution (10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.5) containing DL-P5C (50 μM) 21 and different combinations of NADH (30 μM), 43 NAD + (200 μM), 44 NADPH (40 μM), 45 NADP + (4 μM), 22 and l -proline (100 μM) in a total reaction volume of 1 mL. Reactions were initiated by adding enzyme (0.5 μM final concentration for PYCR1 wild-type and T171M), and initial velocities ( v 0 , μM s –1 ) were determined by monitoring the absorbance decrease at 340 nm.…”